DNA-based analytical techniques, especially array based analytical methodologies, have had a tremendous impact on the development of the oligonucleotide production industry, which developed parallel synthesis techniques in order to meet the demand of the biotechnology market. Parallel synthesis allows large numbers of oligonucleotides to be synthesized on a single apparatus in a single day. This process has been optimized to such a level that most of the material, especially shorter oligonucleotides that are designed as PCR primers, can be used without any purification. In fact, many oligonucleotides are used directly in non-purified form, despite advantages that may be achieved by using purified material.
For many analytical techniques, it is desirable to use longer oligonucleotides (e.g., padlock probes of 70 to 100 nucleotides in length), as their specificity for discriminating between different targets is better then that of shorter oligonucleotides. Crude, synthetic oligonucleotides of this length are heavily contaminated by shorter fragments and need to be purified to avoid problems associated with unspecific binding (Jobs et al. Anal. Chem. 74 (1):199-202 (2002)). Failed couplings and/or side reactions can take place during synthesis that can produce non-full-length or incomplete oligonucleotides. In addition, acid-catalyzed depurination can occur, resulting in cleavage of the oligonucleotide backbone during oligonucleotide deprotection. As a consequence, chemical synthesis produces a population of oligonucleotides, which must be purified to obtain the desired oligonucleotide. Thus, a typical synthetic oligonucleotide reaction mixture contains three major components: full-length product, truncated fragments, and oligonucleotides that result from basic cleavage of previously depurinated oligonucleotide fragments. Full-length products may include deleted fragments (e.g., fragments with single (n−1) or two nucleotide deletions (n−2)). Further, the oligonucleotide reaction mixture may be contaminated by products with unwanted double incorporated nucleotide (n+ products), and also by fragments being incompletely or incorrectly deprotected. Impure oligonucleotides, regardless of length, may cause indistinct results, as found in analyses based on mass spectrometry or in some cases (Villadas et al. Anal. Biochem. 300 (1): 101-103(2002)), impure oligonucleotides can prevent obtaining any significant results.
Thus, efficient purification procedures are needed to process the large number of oligonucleotides that can be synthesized. During synthesis, a trityl moiety typically is left on the 5′ end of the oligonucleotide after coupling of the last nucleotide to facilitate purification of full-length oligonucleotides. The trityl moiety, usually dimethoxytrityl (DMTr) or monomethoxytrityl (MMTr), is an acid labile protecting group that has to be finally removed. This deprotection or detritylation is usually performed in solution using 80% aqueous acetic acid or on a cartridge using an aqueous solution of 2% trifluoroacetic acid (TFA). During detritylation of an oligonucleotide using a cartridge, both the liberated oligonucleotide with free 5′ OH and the newly formed trityl cation must be tightly bound to the cartridge throughout the process. Subsequently, the acid is washed out and detritylated oligonucleotide is eluted with acetonitrile and water. While this procedure appears very straightforward, the detritylation reaction
is reversible and the equilibrium constant depends on the concentration of acid and both products of deprotection. The limited mobility of oligonucleotide and trityl cation on the cartridge results in a very high effective concentration of both of these molecules. As a result, detritylation of oligonucleotides on cartridges can result in low yields of detritylated oligonucleotide (50% or less) and can be accompanied with reassociation of the trityl moiety. Non-detritylated material can be re-detritylated, although the extended deprotection in acid can result in additional apurinic sites in the oligonucleotide. Therefore, a need exists for an efficient method for detritylating oligonucleotides on cartridges.